Functional Expression of Schizosaccharomyces pombe Vba2p in the Vacuolar Membrane of Saccharomyces cerevisiae

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.     

Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Vba4p,a vacuolar membrane protein,is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.     

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomycespombe

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids.     

Isolation from Cigar Tobacco Leaves of Tetrahydroactinidiolide

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.     

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA³ was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Atg22p, a vacuolar membrane protein involved in the amino acid compartmentalization of Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22Δ cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.     

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3^(?1–270) expressed in S. pombe avt3? cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3⁺ but was not completely rescued by the expression of avt3^(?1–270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.     

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3 ⁺-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3 ⁺ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3 ⁺-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.     

Isolation and Characterization of Strains Defective in Vacuolar Ornithine Permease inNeurospora crassa

Vacuolar uptake of ornithine and lysine was characterized inNeurospora crassausing a cupric ion permeabilization system. Michaelis constants were measured as 1.4 mM for lysine and 11.0 mM for ornithine, and maximal velocities were determined. Vacuolar lysine uptake was shown to be inhibited competitively byl-arginine and histidine while ornithine uptake was inhibited by a variety of amino acids. Strains defective in the vacuolar ornithine permease were isolated using a filtration enrichment method. Two isolates—RSC-39 and RSC-63—had a reduced ability to accumulate ornithine. Vacuolar uptake of amino acids was measured using cupric ion-permeabilized mycelia; both strains had reduced ornithine uptake while lysine uptake and arginine uptake were normal. For both isolates, both the Michaelis constant and the maximal velocity for ornithine uptake were reduced compared to those of wild type. These results suggest that both strains are defective in the gene which encodes the vacuolar ornithine permease.     

The Tsc/Rheb signaling pathway controls basic amino acid uptake via the Cat1 permease in fission yeast

The Tsc/Rheb signaling pathway plays critical roles in the control of growth and cell cycle. Studies in fission yeast have also implicated its importance in the regulation of amino acid uptake. Disruption of tsc2 ⁺, one of the tsc ⁺ genes, has been shown to result in decreased arginine uptake and resistance to canavanine. A similar effect is also seen with other basic amino acids. We have identified a permease responsible for the uptake of basic amino acids by genetic complementation and disruption. SPAC869.11 (termed Cat1 for cationic amino acid transporter) contains 12 predicted transmembrane domains and its overexpression in wild type fission yeast leads to the increased uptake of basic amino acids and sensitivity to canavanine. Disruption of cat1 ⁺ in the Δtsc2 background interfered with the suppression of the canavanine-resistant phenotype of Δtsc2 mutants by a dominant negative Rheb. In Δtsc2 mutant strains, the amount of Cat1 was not altered, but instead was mislocalized. This mislocalization was suppressed by the expression of dominant negative Rheb. In addition, we found that the loss of the E3 ubiquitin ligase, Pub1, also restores proper localization. These results provide a crucial link between Tsc/Rheb signaling and the regulation of the basic amino acid permease in fission yeast.     

Expression of a VEGF-like Protein from Parapoxvirus ovis in the Yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S. cerevisiae. Replacement of K2 by an arginine results in stabilization of the protein. This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation. Interestingly, in S. pombe the lysine (K2) has no influence on the stability of the protein. This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways.     

Lysine transport in two barley mutants with altered uptake of basic amino acids in the root

Amino acid uptake was examined in two barley (Hordeum vulgare L.) mutants R906 and R4402 which had been selected as resistant to the lysine analog S-(2-aminoethyl)-cysteine. The mutants were found to be allelic by crossing and examination of F₁ and F₂ progeny. The mutant genes were designated aec1a and aec1b, respectively. The uptake of the basic amino acids lysine, arginine, and ornithine from 50 micromolar solutions was strongly decreased in roots of the mutants, whereas uptake of neutral and acidic amino acids was unaffected. The pattern of uptake of lysine over the range 10⁻⁷ to 10⁻² molar was consistent with there being, principally, two uptake systems operating for basic amino acids in roots and that a low-concentration, high-affinity system is reduced or lacking in the mutants. The residual transport activity in the mutants had a different relative affinity for lysine and arginine to the wild-type system. Uptake of lysine by leaf slices was unimpaired in the mutants suggesting that the leaf uptake system is unaffected by the aec1 gene.     

Peculiarities of amino acid transport inSchizosaccharomyces pombe: Effects of growth medium

Transport systems for amino acids in the wild-type strain ofSchizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used.l-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

Polyphosphate-cation interaction in the amino acid-containing vacuole of Neurospora crassa

The vacuoles of Neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-P. Vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. Thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. Polyphosphate was found to be the only macromolecular polyanion in vacuoles of normal or phosphate-depleted cells. Gel filtration experiments revealed that about half the polyphosphate of normal vacuoles is bound strongly by vacuolar spermidine, Mg2+, and Ca2+. The polyphosphate thus occupied was not available for basic amino acid binding. We have identified about 90% of the cations of isolated vacuoles; in addition to spermidine, Mg2+, and Ca2+, the cation pool consists mainly of arginine, ornithine, histidine, lysine, and Na+, with a small amount of K+. Isolated vacuoles appear to be almost wholly impermeable to all these ions, and in vivo, vacuoles appear to be highly selective in ion uptake by an active process. The interaction of basic amino acid with the available polyphosphate was found to reduce the chemical activity of the former. In keeping with this effect, cells with abnormally high basic amino acid-polyphosphate ratios displayed greatly swollen vacuoles, indicating considerable osmotic activity of the basic amino acids and their counterions under these conditions.     

Characterization of Vacuolar Arginine Uptake and Amino Acid Efflux inNeurospora crassaUsing Cupric Ion to Permeabilize the Plasma Membrane

Treatment ofNeurospora crassamycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K⁺-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: theK_mfor arginine uptake was found to be 4.2–4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.